结核分枝杆菌耐药分子机制及耐药基因检测的新进展

近年来由于结核分枝杆菌(MTB)多重耐药株的蔓延,结核病的发病率在世界范围内呈上升趋势,成为严重的公共卫生问题.进一步了解MTB耐药的分子机制,建立快速、敏感、特异的多重耐药基因检测方法,成为有效监测、控制结核病的必要手段.此文就近几年来这些方面的研究进展进行综述.     

青海地区结核分枝杆菌快速检测及耐药特征研究

目的探讨青海地区耐药结核分枝杆菌(MTB)的相关基因型特点,为开展耐药结核病快速诊断提供依据。方法选择2011年6月-2012年12月西宁市区、农村和农牧区结核病患者的MTB标本各300份,采用MTB微量最低抑菌浓度(MIC)法对耐药结核病进行快速诊断,同时用传统的罗氏药敏法作为对照,采用测序方法分析耐药MTB在不同人群中的分布率,了解青海地区MTB耐药的基因突变特征,基因突变分布和主要流行突变位点。结果西宁市区与农牧区单一耐药及多耐药MTB耐药率比较,牧区明显高于西宁市区和农村,差异有统计学意义(P<0.05);rpoB、katG、gyrA突变率西宁市区分别为52.83%、52.83%、37.74%,农村分别为70.97%、62.90%、45.16%,牧区分别为92.55%、86.17%、81.91%,牧区的基因突变率与西宁市区、农村比较,差异有统计学意义(P<0.05);利福平最常见的突变位点为drpoB(531)占43.66%,异烟肼最常见的突变位点为katG(315),占61.27%;氧氟沙星最常见的突变位点为gyrA占59.52%。结论对青海地区耐药结核分枝杆菌的耐药特征有了进一步了解,为临床医师治疗结核病合理选药提供了依据。     

Molecular detection methods of resistance to antituberculosis drugs in Mycobacterium tuberculosis

Objective

Molecular methods predict drug resistance several weeks before phenotypic methods and enable rapid implementation of appropriate therapeutic treatment. We aimed to detail the most representative molecular tools used in routine practice for the rapid detection of resistance to antituberculosis drugs among Mycobacterium tuberculosis strains.

First insights into circulating Mycobacterium tuberculosis complex lineages and drug resistance in Guinea

In this study we assessed first-line anti-tuberculosis drug resistance and the genotypic distribution of Mycobacterium tuberculosis complex (MTBC) isolates that had been collected from consecutive new tuberculosis patients enrolled in two clinical trials conducted in Guinea between 2005 and 2010. Among the total 359 MTBC strains that were analyzed in this study, 22.8% were resistant to at least one of the first line anti-tuberculosis drugs, including 2.5% multidrug resistance and 17.5% isoniazid resistance, with or without other drugs. In addition, further characterization of isolates from a subset of the two trials (n = 184) revealed a total of 80 different spoligotype patterns, 29 “orphan” and 51 shared patterns. We identified the six major MTBC lineages of human relevance, with predominance of the Euro-American lineage. In total, 132 (71.7%) of the strains were genotypically clustered, and further analysis (using the DESTUS model) suggesting significantly faster spread of LAM10_CAM family (p = 0.00016). In conclusion, our findings provide a first insight into drug resistance and the population structure of the MTBC in Guinea, with relevance for public health scientists in tuberculosis control programs.     

基因芯片技术对结核分枝杆菌耐药性检测的临床应用

目的评价基因芯片技术对结核分枝杆菌耐药性检测的效果。方法选取我院结核科2014年3月至2016年3月收治的涂片阳性的肺结核住院患者238例,取其痰标本,分别用基因芯片、传统的罗氏培养和药敏试验3种方法进行结核分枝杆菌的异烟肼耐药性检测和利福平耐药性检测;把罗氏培养和药敏试验的结果作为金标准,评价基因芯片技术的临床应用价值。结果基因芯片技术检测涂片阳性患者痰标本异烟肼耐药的情况与金标准无显著差异(P>0.05);利福平耐药的情况与金标准相比也无明显差异(P>0.05)。结论基因芯片能够快速、准确地检测结核分枝杆菌对利福平和异烟肼的耐药情况,是一种值得推广的临床实验室检测方法。     

Molecular mechanisms of multidrug resistance in Mycobacterium tuberculosis

Control of tuberculosis caused by multidrug-resistant (MDR) Mycobacterium (M.) tuberculosis has become one of the major problems throughout the world. Understanding of the molecular mechanisms of resistance may help in the development of novel methods for the rapid and precise detection of drug-resistant M. tuberculosis. Eight agents have been recommended to treat tuberculosis. Isoniazid (INH), rifampicin (RFP), pyrazinamide (PZA), streptomycin(SM), and ethambutol (EB) are used as the first line agents, and the others are the second line agents. MDR M. tuberculosis strains are resistant both to INH and RFP which have the most effective bactericidal activity to M. tuberculosis. Nearly 95% of RFP resistant strains possess a mutation on the rpoB gene encoding a DNA-dependent RNA polymerase. INH particularly shows an inhibition of the cell wall synthesis of M. tuberculosis and approximately 90% of INH resistant strains have a mutation on the inhA, katG, and ahpG gene encoding enzymes related to a mycolic acid synthesis of cell wall. PZA resistant strains have a mutation on the pncA gene encoding a pyrazinamidase which degradates pyrazinamide to a bactericidal substance, pyrazinoic acid. SM resistant strains have a mutation on the rrs and rpsL gene encoding a 16S rRNA and a S12 ribosomal subunit protein, respectively. EB resistant strains have a mutation on the embB gene encoding a arabinosyl transferase which catalyzes cell wall synthesis. Resistant mechanisms of second-line agents have also been identified. Recently, rapid detection methods for RFP and INH resistant mutations have been developed on the basis of these studies.     

Genotypes of Mycobacterium tuberculosis in patients at risk of drug resistance in Bolivia

Bolivia ranks among the 10 Latin American countries with the highest rates of tuberculosis (TB) and multidrug resistant (MDR) TB. In view of this, and of the lacking information on the population structure of Mycobacterium tuberculosis in the country, we explored genotype associations with drug resistance and clustering by analyzing isolates collected in 2010 from 100 consecutive TB patients at risk of drug resistance in seven of the nine departments in which Bolivia is divided. Fourteen isolates were MDR, 29 had other drug resistance profiles, and 57 were pansusceptible. Spoligotype family distribution was: Haarlem 39.4%, LAM 26.3%, T 22.2%, S 2.0%, X 1.0%, orphan 9.1%, with very low intra-family diversity and absence of Beijing genotypes. We found 66 different MIRU-VNTR patterns; the most frequent corresponded to Multiple Locus Variable Analysis (MLVA) MtbC15 patterns 860, 372 and 873. Twelve clusters, each with identical MIRU-VNTR and spoligotypes, gathered 35 patients. We found no association of genotype with drug resistant or MDR-TB. Clustering associated with SIT 50 and the H3 subfamily to which it belongs (p < 0.0001). The largest cluster involved isolates from three departments and displayed a genotype (SIT 50/MLVA 860) previously identified in Bolivian migrants into Spain and Argentina suggesting that this genotype is widespread among Bolivian patients. Our study presents a first overview of M. tuberculosis genotypes at risk of drug resistance circulating in Bolivia. However, results should be taken cautiously because the sample is small and includes a particular subset of M. tuberculosis population.     

Four year longitudinal study of Mycobacterium tuberculosis complex isolates in a region of North-Eastern Italy

Recent reports have suggested a change of Mycobacterium tuberculosis complex genetic diversity in Western Europe due to an increasing proportion of imported cases of tuberculosis (TB). This study analyzed a total of 705 M. tuberculosis strains isolated from 2006 to 2009 in Veneto, a North-Eastern Italian region, to see the impact of foreign-born cases vs. Italian patients on prevailing TB epidemiology. Strains were genotyped using spoligotyping followed by comparison with international genotyping database SITVIT2. Six spoligotyping clusters with suspected phylogeographical specificity for imported cases, were typed by 15-loci MIRUs for a finer characterization. Overall, 410 (58.16%) strains were isolated from foreign-born patients, while 295 (41.84%) were isolated from Italian patients. Older patients (>70 years, i.e., 46.4% of cases) predominated among Italians while younger age groups prevailed among foreign-born patients. Our results suggest that despite a high proportion of reactivation of latent TB infection in elderly Italian-born patients, active TB transmission between foreign-born and Italian patients may be ongoing, and argue in favor of an increased TB surveillance among immigrants to combat TB epidemic in Italy.     

结核分枝杆菌耐多药情况分析

目的：评估上海市近年来耐多药结核病（MDR—TB）发生情况。方法：对本市2749株结核分枝杆菌耐药性测定结果进行分析。结果：共查出234株耐多药菌株，其中新发病。组156株（6．8％），复治病人组35株（18．9％），外地病人组43株（15．8％）。234株耐多药菌株中，耐2药133株（56．8％），耐3药66株（28．2％），耐4药35株（15．0％）。耐2药菌株中，以HR耐药为主，占27．8％。耐3药菌株中，以HRS耐药为主，占66．7％。复治病人和外地病人组的耐多药率显著高于新发病人组，P＜0．01。结论：必须加强对复治病人和外地病人的管理和治疗。     

Use of applied theatre in health research dissemination and data validation: a pilot study from South Africa

This article reports on a pilot study of the use of applied theatre in the dissemination of health research findings and validation of data. The study took place in South Africa, as part of the Southern Africa Stroke Prevention Initiative (SASPI) and was based at the University/Medical Research Council Rural Public Health and Health Transitions Research Unit (also known as the Agincourt Unit). The aim of SASPI was to investigate the prevalence of stroke and understand the social context of stroke. It was decided to use an applied theatre approach for validating the data and disseminating findings from the anthropological component of the study. The pilot study found that applied theatre worked better in smaller community groups. It allowed data validation and it elicited ideas for future interventions resulting from the health research findings. Evaluation methods of the impact of applied theatre as a vehicle for the dissemination and communication of research findings require further development.     

Molecular typing and drug susceptibility of Mycobacterium tuberculosis isolates from Chongqing Municipality,China

China’s tuberculosis (TB) burden is second only to that of India worldwide. In Chongqing, the largest municipality in southwestern China, although the prevalence of both TB and drug-resistant TB is higher than in other municipalities, the molecular characteristics and drug susceptibility phenotypes are poorly known. In this study, 297 Mycobacterium tuberculosis isolates from Chongqing were genotyped with spacer oligonucleotide typing (spoligotyping) and 28-locus MIRU-VNTR (24-locus MIRU-VNTR scheme and 4 other loci). Spoligotyping results were compared with drug-resistant profiles. Patients who showed clustering by both spoligotyping and 28-locus MIRU-VNTR were interviewed to investigate their detailed contact history. Our data demonstrated that the Beijing genotype was the most prevalent genotype, and ST1 was the most predominant lineage in Chongqing. The Beijing genotype was significantly associated with ethambutol resistance and multidrug-resistant phenotypes. A combination of the 10 most polymorphic loci permitted to achieve higher discriminatory power than 24-VNTR. In addition, a presumed transmission pathway was observed in a cluster of patients with the same MIRU-VNTR profile. The 10-VNTR locus set is suitable for genotyping of Mycobacterium tuberculosis in Chongqing.     

202例肺结核患者痰培养阳性菌株耐药性分析

目的了解某市胸科医院住院患者痰标本分离结核分枝杆菌的耐药情况。方法回顾性调查该院2003年1月1日-2006年12月31日所有新发和复治的肺结核病患者痰标本分离结核分枝杆菌对异烟肼、链霉素、乙胺丁醇和利福平的敏感性。结果4年间202例住院肺结核患者痰标本分离的结核分枝杆菌总耐药率为39.11%（79/202）,总耐多药率为10.89%（22/202）。初始耐药率为35.11%,获得性耐药率为46.48%。获得性耐多药率16.90%,明显高于初始耐多药率7.63%（χ2=4.08,P＝0.049）。2004年度获得性耐多药率36.36%,明显高于初始耐多药率5.66%（χ2=5.95,P=0.02）;2006年获得性耐多药率与初始耐多药率相同,均为13.51%。复治患者对异烟肼、链霉素、利福平均显示较高的耐药性,耐药率分别为47.89%、46.48%、36.62%,复治患者耐多药率为35.21%;初治患者单耐药率和多耐药率均为16.03%。结论耐药和耐多药肺结核病疫情严重,特别是初始耐药和耐多药结核病,故有效控制耐药结核病是我们当前所面临的紧迫任务。     

兰州地区结核分支杆菌耐药性分析

目的 分析兰州地区2005～2007年结核分支杆菌耐药情况,为结核病的防治提供科学依据.方法 利用比例法对215株菌株进行药物敏感性检测,用SPSS 11.0进行统计分析.结果 兰州地区结核分支杆菌总耐药率为47.51％,耐多药率4.75％,低于2001～2004年统计结果,原发性耐药占耐药菌株总数的55％,获得性耐药率为46.59％.结论 兰州地区结核病患者结核分枝杆菌耐药率有所下降,但结核病传染源的控制、结核病防治宣教及结核病短程化疗(DOTS)健康教育和促进工作都需进一步加强.     

Contribution of efflux activity to isoniazid resistance in the Mycobacterium tuberculosis complex

Resistance to isoniazid (INH), one of the main drugs used in tuberculosis (TB) therapy, is mostly due to chromosomal mutations in target genes. However, approximately 20–30% of INH resistant Mycobacterium tuberculosis isolates do not have mutations in any of the genes associated with INH resistance. This suggests that other mechanism(s) may be involved, namely efflux pump systems capable of extruding the drug to the exterior of the cell. In a previous work, we have induced clinical INH susceptible M. tuberculosis isolates and the H37Rv reference strain to high-level resistance to INH, by gradual exposure to increasing concentrations of this drug. In the present study, we have characterized these strains and Mycobacterium bovis BCG induced to INH resistance with respect to their efflux activity and its contribution to INH resistance using the following approach: determination of the susceptibility to INH in the presence and absence of the efflux inhibitors (EIs) chlorpromazine, thioridazine and verapamil; evaluation of efflux activity by a semi-automated fluorometric method; and quantification of the expression level of genes coding for efflux pumps by real-time RT-qPCR.The EIs decreased INH resistance in the INH induced strains, in particular verapamil promoted a reversal of resistance in some of the strains tested. The induced strains presented an increased efflux activity that was inhibited by the EIs and showed overexpression of the efflux pump genes efpA, mmpL7, mmr, p55 and the Tap-like gene Rv1258c. Altogether, these results correlate efflux activity with INH resistance and demonstrate that efflux pumps play an important role in acquired INH resistance in M. tuberculosis complex. The development of EIs that can restore the antimicrobial activity of the antibiotic subject to efflux is an approach that can be useful in order to prevent the emergence of this resistance and guide the development of new effective anti-TB therapeutical approaches.     

结核分枝杆菌耐药表型与耐药基因型相关性研究进展

20世纪80年代以来,结核病（tuberculosis,TB）疫情呈全球性回升趋势,我国作为全球最大的发展中国家,同时也是全球TB疫情最严重的国家之一［1］。随着全球耐药结核病（drug resistant tuberculosis,DR TB）的出现和传播,特别是耐多药结核病（multidrug resistant TB,MDR TB）、广泛耐药结核病（extensively drug resistant TB,XDR TB）,以及全耐药结核病（totally drug resistant TB,TDR TB）发生率的增高,全球TB的有效治疗和控制受到严重威胁。深入研究结核分枝杆菌（Mycobacterium tuberculosis,MTB）的耐药分子机制,发现基因突变是MTB产生耐药的重要原因。现就各抗结核药物的耐药表型与耐药基因型的相关性研究进行综述。     

Quadruple-first line drug resistance in Mycobacterium tuberculosis in Vietnam: What can we learn from genes?

In Vietnam, a country with high tuberculosis (137/100.000 population) and multidrug-resistant (MDR)-TB burdens (7.8/100.000 population), little is known about the molecular signatures of drug resistance in general and more particularly of second line drug (SLD) resistance. This study is specifically focused on Mycobacterium tuberculosis isolates resistant to four first-line drugs (FLDs) that make TB much more difficult to treat. The aim is to determine the proportion of SLD resistance in these quadruple drug resistant isolates and the genetic determinants linked to drug resistance to better understand the genetic processes leading to quadruple and extremely drug resistance (XDR). 91 quadruple (rifampicin, isoniazid, ethambutol and streptomycin) FLD resistant and 55 susceptible isolates were included. Spoligotyping and 24-locus MIRU-VNTR techniques were performed and 9 genes and promoters linked to FLD and SLD resistance were sequenced. SLD susceptibility testing was carried out on a subsample of isolates. High proportion of quadruple-FLD resistant isolates was resistant to fluoroquinolones (27%) and second-line injectable drugs (30.2%) by drug susceptibility testing. The sequencing revealed high mutation diversity with prevailing mutations at positions katG315, inhA-15, rpoB531, embB306, rrs1401, rpsL43 and gyrA94. The sensitivity and specificity were high for most drug resistances (> 86%), but the sensitivity was lower for injectable drug resistances (< 69%). The mutation patterns revealed 23.1% of pre-XDR and 7.7% of XDR isolates, mostly belonging to Beijing family. The genotypic diversity and the variety of mutations reflect the existence of various evolutionary paths leading to FLD and SLD resistance. Nevertheless, particular mutation patterns linked to high-level resistance and low fitness costs seem to be favored.     

2015-2018年陕西某医院结核分枝杆菌耐药趋势分析

目的分析本院结核分枝杆菌的耐药情况及趋势,为耐药结核病的预防及治疗提供参考依据。方法对2015-2018年本院门诊、住院病人,共9050例结核分枝杆菌的一线和二线抗结核药物耐药情况进行回顾性分析。结果:四年中,本院结核菌培养的阳性率有所上升,但是总体耐药率基本不变(平均35.03%)。单耐药的耐药率有所下降(X²=13.76,P=0.003),而耐多药和一线4种耐药的耐药率有所上升(耐多药X²=19.84,P<0.001;一线4种耐药X²=18.97,P<0.001)。异烟肼、链霉素、乙胺丁醇、利福平、阿米卡星和丙硫异烟胺的耐药率都有所上升(分别为X²=12.57,P=0.006;X²=17.02,P=0.001;X²=13.68,P=0.003;X²=25.60,P<0.001;X²=12.82,P=0.005;X²=11.19,P=0.011)。结论单耐药减少,但耐多药...     

Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment.

The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.     

Molecular detection and characterization of novel haemotropic Mycoplasma in free-living mole rats from South Africa

The importance of haemotropic Mycoplasma (haemoplasma) infections to animal and human health is increasingly recognised. Although wild rodents are known to host these bacteria, haemoplasma prevalence and diversity in small mammals is under-documented, globally. This is due to the reliance on molecular approaches to detect these unculturable, obligate bacteria and to a paucity of assays targeting informative gene regions. We attempted to address these challenges by evaluating the performance of three 16S rRNA PCR assays for detecting Mycoplasma in four African mole-rat species of the family Bathyergidae. This was achieved by screening DNA samples prepared from lung and liver samples of 260 bathyergids, sampled from natural and urban landscapes in the Western Cape Province with one published and two novel conventional PCR assays. Sequence-confirmed Mycoplasma presence guided calculations of the relative sensitivity and specificity of the assays and revealed that 26.5% of the rodents were haemoplasma-positive. Bathyergus suillus sampled near an informal human settlement had a significantly higher infection rate (42%) than the three bathyergid species sampled from natural settings, for which PCR-positivity ranged from 0% to 36%. The 16S rRNA gene phylogeny identified the presence of six Mycoplasma strains in bathyergids that form a novel monophyletic lineage belonging to the haemofelis group, with 16S rRNA and Rnase P gene phylogenies indicating that the bathyergid-associated haemoplasmas were novel and closely related to Mycoplasma coccoides. Assay sensitivity ranged from 60.3% to 76.8% and specificity from 94.8% to 100% and both were highest for the novel assay targeting a ~ 300 bp region of the 16S rRNA gene. Results confirm the presence of novel haemoplasma strains in bathyergid species from South Africa and emphasise the need for expanded studies on haemoplama prevalence, diversity, and transmission routes in other small mammal species from this biodiverse region.